This project investigates the hypothesis that fibrillar collagen content is the principle matrix characteristic limiting interstitial diffusion of gene vector therapeutics in tumors. The research proposed herein will provide much needed information on the ability of vectors to diffuse in the tumor interstitium and will explore the influence of matrix collagen content on this diffusion. These results could be used to guide drug design and to identify matrix-modifying treatments to improve drug delivery. The project is divided into four aims. First, our existing fluorescence recovery after photobleaching (FRAP) setup will be converted to a photoactivated fluorescence (PAF) to allow the study of large molecule diffusion in vitro and in vivo. Second, PAF will be used to measure diffusion of vectors in collagen gels to allow correlation between collagen content and diffusion coefficients in vitro. Third, collagen content and diffusion coefficients of the same vectors will be measured in human tumor xenografts implanted in dorsal skin fold chambers of immunodeficient mice; implanted tumors will be of types LS174T, a low collagen content carcinoma, and U87, a high collagen content glioma. Finally, the diffusion coefficients and collagen content will be measured again after the tumors are treated with collagenase to break down collagen. All data will be analyzed for a correlation between diffusion coefficient and collagen content, and diffusion in collagen gels and in tumors will be compared. Diffusion coefficients in collagenase treated tumors will be compared with those in untreated tumors to determine if collagen-reducing treatment may improve drug delivery.